GSM6045745: Neuron elav INTACT Lola I 14-17h 1; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
lola
Cell type
Cell type Class
Embryo
Cell type
14-17h embryos
NA
NA
Attributes by original data submitter
Sample
source_name
Lola-I ChIP in 14-17hrs INTACT embryos in Neuron Rep1
tissue
Neuron
chip antibody
anti-Lola-I rabbit polyclonal, custom (GenScript)
developmental stage
14-17h
genotype
wildtype
geo_loc_name
missing
collection_date
missing
Sequenced DNA Library
library_name
GSM6045745
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Nuclei were isolated from INTACT embryos 14-17h AED by resuspending and douncing embryos in HBS buffer (0.125 M Sucrose, 15 mM Tris (pH 7.5), 15 mM NaCl, 40 mM KCl, 2 mM EDTA, 0.5 mM EGTA). The nuclei were further dissociated using syringe (22.5 gauge needle) 10 times. After washins with HBS buffer, nuclei were incubated with Dynabeads® M-280 Streptavidin beads( Invitrogen, # 11205D) for 30 minutes with end to end rotation at 4°C. A magnet was used to separate the bead bound nuclei, and the beads were washed thoroughly with HBS buffer. Purity of nuclei isolation was determined by staining nuclei with DAPI, and presence of free nuclei, unbound to beads, were used as indicators of contamination. Isolated nuclei were used for ChIP-seq. ChIP-seq was performed as follows. ~100 mg embryos were used per ChIP, and 5 µg chromatin was used for tissue-specific ChIP-seq experiments. Fixed embryos were homogenized by douncing in an ice cold A1 buffer (15 mM HEPES (pH 7.5), 15 mM NaCl, 60 mM KCl, 4 mM MgCl, 0.5% Triton X-100, 0.5mM DTT, protease inhibitors) and A2 buffer (15 mM HEPES (pH 7.5), 140 mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, protease inhibitors) in a tissue grinder for 10-15 times in A1 and A2 buffer each. Then the sonication of the chromatin was performed with a Bioruptor Pico for four-five rounds of 30 s on and 30 s off cycles. The sonicated chromatin was cleared by centrifugation and the supernatant was used for ChIP. Chromatin was incubated with antibodies pre-bound to Dynal magnetic beads (IgA or IgG) overnight with end-to-end rotation at 4°C and washed with an ice cold RIPA buffer (50 mM HEPES (pH 7.5), 1mM EDTA, 0.7% sodium deoxycholate, 1% NP-40 (IGEPAL CA-630), 0.5M LiCl). Eluted, reverse cross-linked DNA was then purified using phenol-chloroform-isoamylalcohol phase separation and ethanol precipitation. ChIP-seq libraries were prepared from 5-15 ng ChIP DNA or 100 ng input DNA according to the manufacturer's instructions (NEBNext ChIP-Seq Library Prep kit).